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WO1992003545A1 - Vaccin a base de poxvirus recombine contre le flavivirus - Google Patents

Vaccin a base de poxvirus recombine contre le flavivirus Download PDF

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Publication number
WO1992003545A1
WO1992003545A1 PCT/US1991/005816 US9105816W WO9203545A1 WO 1992003545 A1 WO1992003545 A1 WO 1992003545A1 US 9105816 W US9105816 W US 9105816W WO 9203545 A1 WO9203545 A1 WO 9203545A1
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WO
WIPO (PCT)
Prior art keywords
virus
recombinant
vaccinia
plasmid
fragment
Prior art date
Application number
PCT/US1991/005816
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English (en)
Inventor
Enzo Paoletti
Steven Elliot Pincus
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Virogenetics Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US07/714,687 external-priority patent/US5514375A/en
Application filed by Virogenetics Corporation filed Critical Virogenetics Corporation
Priority to AU87287/91A priority Critical patent/AU657711B2/en
Priority to JP51661991A priority patent/JP3955315B2/ja
Priority to GB9303023A priority patent/GB2269820B/en
Publication of WO1992003545A1 publication Critical patent/WO1992003545A1/fr

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Definitions

  • the present invention relates to a modified poxvirus and to methods of making and using the same. More in particular, the invention relates to recombinant poxvirus, which virus expresses gene products of a flavivirus gene, and to vaccines which provide protective immunity against flavivirus infections.
  • Several publications are referenced in this application. Full citation to these references is found at the end of the specification preceding the claims. These references describe the state-of-the-art to which this invention pertains. BACKGROUND OF THE INVENTION
  • Vaccinia virus and more recently other poxviruses have been used for the insertion and expression of foreign genes.
  • the basic technique of inserting foreign genes into live infectious poxvirus involves recombination between pox DNA sequences flanking a foreign genetic element in a donor plasmid and homologous sequences present in the rescuing poxvirus (Piccini et al., 1987).
  • the recombinant poxviruses are constructed in two steps known in the art and analogous to the methods for creating synthetic recombinants of the vaccinia virus described in U.S. Patent No. 4,603,112, the disclosure of which patent is incorporated herein by reference.
  • the DNA gene sequence to be inserted into the virus is placed into an E . coli plasmid construct into which DNA homologous to a section of DNA of the poxvirus has been inserted.
  • the DNA gene sequence to be inserted is ligated to a promoter.
  • the promoter-gene linkage is positioned in the plasmid construct so that the promoter-gene linkage is flanked on both ends by DNA homologous to a DNA sequence flanking a region of pox DNA containing a nonessential locus.
  • the resulting plasmid construct is then amplified by growth within E.
  • the isolated plasmid containing the DNA gene sequence to be inserted is transfected into a cell culture, e.g. chick embryo fibroblasts, along with the poxvirus. Recombination between homologous pox DNA in the plasmid and the viral genome respectively gives a poxvirus modified by the presence, in a nonessential region of its genome, of foreign DNA sequences.
  • the term "foreign" DNA designates exogenous DNA, particularly DNA from a non-pox source, that codes for gene products not ordinarily produced by the genome into which the exogenous DNA is placed.
  • Genetic recombination is in general the exchange of homologous sections of DNA between two strands of DNA.
  • RNA may replace DNA.
  • Homologous sections of nucleic acid are sections of nucleic acid (DNA or RNA) which have the same sequence of nucleotide bases.
  • Genetic recombination may take place naturally during the replication or manufacture of new viral genomes within the infected host cell. Thus, genetic recombination between viral genes may occur during the viral replication cycle that takes place in a host cell which is co-infected with two or more different viruses or other genetic constructs.
  • a section of DNA from a first genome is used interchangeably in constructing the section of the genome of a second co-infecting virus in which the DNA is homologous with that of the first viral genome.
  • recombination can also take place between sections of DNA in different genomes that are not perfectly homologous. If one such section is from a first genome homologous with a section of another genome except for the presence within the first section of, for example, a genetic marker or a gene coding for an antigenic determinant inserted into a portion of the homologous DNA, recombination can still take place and the products of that recombination are then detectable by the presence of that genetic marker or gene in the recombinant viral genome.
  • Successful expression of the inserted DNA genetic sequence by the modified infectious virus requires two conditions. First, the insertion must be into a nonessential region of the virus in order that the modified virus remain viable.
  • the second condition for expression of inserted DNA is the presence of a promoter in the proper relationship to the inserted DNA. The promoter must be placed so that it is located upstream from the DNA sequence to be expressed.
  • Flaviviridae comprises approximately 60 arthropod-borne viruses that cause significant public health problems in both temperate and tropical regions of the world (Shope, 1980; Monath, 1986) . Although some highly successful inactivated vaccines and live-attenuated vaccines have been developed against some of these agents, there has been a recent surge in the study of the molecular biology of flaviviruses in order to produce recombinant vaccines to the remaining viruses, most notably dengue (Brandt, 1988) .
  • Flavivirus proteins are encoded by a single long translational open reading frame (ORF) present in the positive-strand genomic RNA. The genes encoding the structural proteins are found at .the 5• end of the genome followed by the nonstructural glycoprotein NSl and the remaining nonstructural proteins (Rice et al., 1985).
  • the flavivirus virion contains an envelope glycoprotein, E, a membrane protein, M, and a capsid protein, C.
  • E envelope glycoprotein
  • M a membrane protein
  • C capsid protein
  • virion preparations usually contain a small amount of the glycoprotein precursor to the membrane protein, prM (Mason et al., 1987a). Within JEV-infected cells, on the other hand, the M protein is present almost exclusively as the higher molecular weight prM protein (Mason et al. , 1987a; Shapiro et al. , 1972).
  • Monoclonal antibodies to E can provide protection from infection by Japanese encephalitis virus (JEV) (Kimura-Kuroda et al., 1988; Mason et al., JEV) (Kimura-Kuroda et al., 1988; Mason et al., JEV) (Kimura-Kuroda et al., 1988; Mason et al., JEV) (Kimura-Kuroda et al., 1988; Mason et al.,
  • NSl immunity Additional support for the ability of NSl immunity to protect the host from infection comes from direct immunization experiments in which NSl purified from either yellow fever virus-infected cells (Schlesinger et al., 1985, 1986) or dengue type 2 virus-infected cells (Schlesinger et al., 1987) induced protective immunity from infection with the homologous virus. Although significant progress has been made in deriving the primary structure of these three flavivirus glycoprotein antigens, less is known about their three- dimensional structure. The ability to produce properly folded, and possibly correctly assembled, forms of these antigens may be important for the production of effective recombinant vaccines.
  • E and prM into viral particles may require the coordinated synthesis of the NSl protein, which is coretained in an early compartment of the secretory apparatus along with immature forms of E in JEV-infected cells (Mason, 1989) .
  • Yasuda et al. (1990) reported a vaccinia recombinant containing the region of JEV encoding 65 out of the 127 amino acids of C, all of prM, all of E, and 59 out of the 352 amino acids of NSl.
  • Haishi et al. (1989) reported a vaccinia recombinant containing Japanese encephalitis sequences encoding 17 out of the 167 amino acids of prM, all of E and 57 out of the 352 amino acids of NSl.
  • Zhao et al. (1987) reported a vaccinia recombinant containing the dengue-4 coding sequences for all of C, all of prM, all of E, all of NSl, and all of NS2A. Bray et al.
  • Dengue type 2 structural proteins have been expressed by recombinant vaccinia viruses (Deubel et al., 1988) . Although these viruses induced the synthesis of the structural glycoprotein within infected cells, they neither elicited detectable anti-dengue immune responses nor protected monkeys from dengue infection. Several studies also have been completed on the expression of portions of the dengue type 4 structural and nonstructural proteins in vaccinia virus (Bray et al., 1989; Falgout et al., 1989; Zhao et al., 1987).
  • a dengue-vaccinia recombinant expressing a C-terminally truncated E protein gene induced the synthesis of an extracellular form of E and provided an increasing level of resistance to dengue virus encephalitis in inoculated mice (Men et al., 1991).
  • the present invention relates to a recombinant poxvirus generating an extracellular flavivirus structural protein capable of inducing protective immunity against flavivirus infection.
  • the recombinant poxvirus generates an extracellular particle containing flavivirus E and M proteins capable of eliciting neutralizing antibodies and hemagglutination-inhibiting antibodies.
  • the poxvirus is advantageously a vaccinia virus or an avipox virus, such as fowlpox virus or canarypox virus.
  • the flavivirus is advantageously Japanese encephalitis virus, yellow fever virus and Dengue virus.
  • the recombinant poxvirus contains therein DNA from flavivirus in a nonessential region of the poxvirus genome for expressing in a host flavivirus structural protein capable of release to an extracellular medium.
  • the DNA contains Japanese encephalitis virus coding sequences that encode a precursor to structural protein M, structural protein E, and nonstructural proteins NSl and NS2A.
  • the recombinant poxvirus contains therein DNA from flavivirus in a nonessential region of the poxvirus genome for expressing a particle containing flavivirus structural protein E and structural protein M.
  • the present invention relates to a vaccine for inducing an immunological response in a host animal inoculated with the vaccine, said vaccine including a carrier and a recombinant poxvirus containing, in a nonessential region thereof, DNA from flavivirus.
  • the recombinant viruses express portions of the flavivirus ORF extending from prM to NS2B. Biochemical analysis of cells infected with the recombinant viruses showed that the recombinant viruses specify the production of properly processed forms of all three flavivirus glycoproteins - prM, E, and NSl. The recombinant viruses induced synthesis of extracellular particles that contained fully processed forms of the M and E proteins.
  • FIG. 1 schematically shows a method for the construction of donor plasmids pSPJEVSH12VC and pSPJEVL14VC containing coding sequences for a portion of the JEV structural protein coding region, NSl and NS2A;
  • FIG. 2 schematically shows a method for the construction of donor plasmids pSPJEVUVC and pSPJEVlOVC containing coding sequences for a portion of the JEV structural protein coding region, NSl, NS2A and NS2B;
  • FIG. 3 shows the DNA sequence of oligonucleotides (shown with translational starts and stops in italics and early transcriptional stops underlined) used to construct the donor plasmids;
  • FIG. 4 is a map of the JEV coding regions inserted in the four recombinant vaccinia viruses VP650, vP555, VP658 and VP583;
  • FIG. 8 shows a comparison by SDS-PAGE analysis of the culture fluid E proteins produced by JEV infection and infection with the recombinant vaccinia viruses vP650, VP555, VP658 and vP583;
  • FIG. 10 shows a comparison by immunoprecipitation analysis of the JEV-specific reactivity of the pre-challenge sera from animals vaccinated with JEV and with vaccinia recombinants vP555 and vP658;
  • FIG. 11 schematically shows a method for the construction of plasmid pSD460 for deletion of thymidine kinase gene and generation of recombinant vaccinia virus VP410
  • FIG. 12 schematically shows a method for the construction of plasmid pSD486 for deletion of hemorrhagic region and generation of recombinant vaccinia virus vP553;
  • FIG. 13 schematically shows a method for the construction of plasmid pMP494 ⁇ for deletion of ATI region and generation of recombinant vaccinia virus vP618;
  • FIG. 14 schematically shows a method for the construction of plasmid pSD467 for deletion of hemagglutinin gene and generation of recombinant vaccinia virus VP723;
  • FIG. 15 schematically shows a method for the construction of plasmid pMPCSKl ⁇ for deletion of gene cluster [C7L - K1L] and generation of recombinant vaccinia virus VP804;
  • FIG. 16 schematically shows a method for the construction of plasmid pSD548 for deletion of large subunit, ribonucleotide reductase and generation of recombinant vaccinia virus vP866 (NYVAC) ;
  • FIG. 17 shows the DNA sequence of the Nakayama strain of JEV in the region encoding C through NS2B;
  • FIG. 18 is a map of the JEV coding regions inserted in the vaccinia viruses VP555, vP825, VP908, VP923,
  • FIG. 19 is a map of the YF coding regions inserted in the vaccinia viruses VP766, VP764, VP869, VP729, VP725, vP984, vP997, vP1002, vP1003 and canarypox virus vCP127;
  • FIG. 20 shows part of the DNA sequence of a
  • FIG. 21 is a map of the DEN coding regions inserted in the vaccinia viruses vP867, vP962 and VP955.
  • FIG. 22 shows the DNA sequence of a canarypox
  • FIG. 23 schematically shows a method for the construction of plasmid pRW848 for deletion of C5;
  • FIG. 24 shows the DNA sequence of a 7351 base pair fragment of canarypox containing the C3 ORF.
  • a thy idine kinase mutant of the Copenhagen strain of vaccinia virus, VP410 (Guo et al., 1989), was used to generate recombinant vP658 (see below) .
  • a recombinant vaccinia virus (vP425) containing the Beta-galactosidase gene in the HA region under the control of the 11-kDa late vaccinia virus promoter (Guo et al., 1989) was used to generate recombinants vP555, vP583 and vP650. All vaccinia virus stocks were produced in either VERO (ATCC CCL81) or
  • MRC-5 (ATCC CCL171) cells in Eagle's minimal essential medium (MEM) plus 10% heat-inactivated fetal bovine serum (FBS) .
  • FBS heat-inactivated fetal bovine serum
  • Biosynthetic studies were performed using baby hamster kidney cells (BHK 21-15 clone) grown at 37°C in MEM supplemented with 7.5% FBS and antibiotics, or VERO cells grown under the same conditions except using 5% FBS.
  • the JEV virus used in all in vitro experiments was a clarified culture fluid prepared from C6/36 cells infected with a passage 55 suckling mouse brain suspension of the Nakayama strain of JEV (Mason, 1989) . .
  • Restriction enzymes were obtained from GIBCO/BRL, Inc. , (Gaithersburg, MD) , New England BioLabs, Inc. (Beverly, MA) , or Boehringer Mannheim Biochemicals (Indianapolis, IN) .
  • T4 DNA ligase was obtained from New England BioLabs, Inc. Standard recombinant DNA techniques were used (Maniatis et al., 1986) with minor modifications for cloning, screening, and plasmid purification. Nucleic acid sequences were confirmed using standard dideoxy chain-termination reactions (Sanger et al., 1977) on alkaline-denatured double-stranded plasmid templates.
  • JEV cDNAs used to construct the JEV-vaccinia recombinant viruses were derived from the Nakayama strain of JEV (McAda et al., 1987); all nucleotide coordinates are derived from the sequence data presented in FIG. 17A and B (SEQ ID NO:52) which contains the sequence of the C coding region combined with an updated sequence of prM, E, NSl, NS2A and NS2B coding regions.
  • Plasmid pJEV3/4 was derived by cloning a B ⁇ lll-Apal fragment of JEV cDNA (nucleotides 2554-3558) , an Apal-Ball fragment (nucleotides 3559-4125) , and annealed oligos J3 (SEQ ID NO:44) and J4 (SEQ ID N0:45) [FIG. 3; containing a translation stop followed by a vaccinia early transcription termination signal (TTTTTAT; Yuen et al. , 1987) , an Ea ⁇ l site, and a Hindlll sticky end] into
  • pJEV3/4 was digested within the JEV sequence by EcoRV (nucleotide 2672) and within pUC18 by SacI, and the fragment containing the plasmid origin and JEV cDNA sequences extending from nucleotides 2672-4125 was ligated to a SacI-EcoRV fragment of JEV cDNA (nucleotides 2125-2671) .
  • the resulting plasmid, pJEVl contained the viral ORF extending from the SacI site (nucleotide 2125) in the last third of E through the Ball site (nucleotide 4125) two amino acid residues (aa) into the predicted N terminus of NS2B (FIG. 1) .
  • Synthetic oligos JIB (SEQ ID NO:46) and J2B (SEQ ID NO:47) (FIG. 3; containing a Xhol sticky end, a Smal site, the last 15 aa of C, and first 9 aa of JEV prM with a sticky Hindlll end) were ligated to a Hindlll-SacI fragment of JEV cDNA (nucleotides 407-2124) , and XhoI-SacI digested vector pIBI24 (International Biotechnologies Inc. , New Haven, CT) .
  • the resulting plasmid, pJEV2 contained the viral ORF extending between the methionine (Met) codon (nucleotides 337-339) occurring 15 aa preceding the predicted N terminus of prM and the SacI site (nucleotide 2124) found in the last third of E (FIG. 1).
  • pTP15 contains the early/late vaccinia virus H6 promoter inserted into a polylinker region flanked by sequences from the Hindlll A fragment of vaccinia virus from which the hemagglutinin (HA) gene has been deleted (Guo et al., 1989) .
  • Smal-Eagl digested pTP15 was purified and ligated to the purified Smal-SacI insert from pJEV2 plus the SacI-EagI insert of pJEVl, yielding pSPJEVL (FIG. 1) .
  • the Smal-EagI pTP15 fragment was ligated to the purified Smal-SacI insert from pJEV5 plus the SacI-EagI insert of pJEVl, yielding pSPJEVSH (FIG. 1) .
  • the 6 bp corresponding to the unique Smal site used to produce pSPJEVSH were removed as described above, creating pSPJEVSH12VC in which the H6 promoter immediately preceded the ATG start codon (FIG. 1) .
  • Oligonucleotide-directed mutagenesis (Kunkel,
  • Synthetic oligos J37 and J38 [FIG. 3; containing JEV nucleotides 4497-4512, a translation stop, an early transcription termination signal (TTTTTAT; Yuen et al., 1987) , an Eacrl site, and Hindlll sticky end] were used to clone a Sacl-Dral fragment of JEV cDNA (nucleotides
  • pJEV7 contained the viral ORF extending between the SacI site (nucleotide 2125) found in the last third of E and the last codon of NS2B (nucleotide 4512) (FIG. 2) .
  • Smal-EagI digested pTP15 was purified and ligated to the purified Smal-SacI insert from pJEV22 plus the SacI-EagI insert of pJEV7, yielding pSPJEVIO (FIG. 2).
  • Example 3 - JN VITRO VIRUS INFECTION AND RADIOLABELING BHK or VERO cell monolayers were prepared in 35 mm diameter dishes and infected with vaccinia viruses (m.o.i. of 2) or JEV (m.o.i. of 5) and incubated for 11 hr (vaccinia) or 16 hr (JEV) before radiolabeling. At 11 hr or 16 hr post-infection, the medium was removed and replaced with warm Met-free medium containing 2% FBS and 250 ⁇ Ci/ml of 35 S-Met.
  • the cells were incubated for 1 hr at 37°C, rinsed with warm maintenance medium containing 10-times the normal amount of unlabeled Met, and incubated in this same high Met medium 6 hr before harvesting as described below.
  • samples of clarified culture fluid were analyzed by sucrose gradient centrifugation in 10 to 35% continuous sucrose gradients prepared, centrifuged, and analyzed as described (Mason, 1989) .
  • Radiolabeled cell lysates and culture fluids were harvested and the viral proteins were immunoprecipitated, digested with endoglycosidases, and separated in
  • vaccinia virus recombinants Four different vaccinia virus recombinants were constructed that expressed portions of the JEV coding region extending from prM through NS2B.
  • the JEV cDNA sequences contained in these recombinant viruses are shown in FIG. 4.
  • the sense strand of the JEV cDNA was positioned behind the vaccinia virus early/late H6 promoter, and translation was expected to be initiated from naturally occurring JEV Met codons located at the 5' ends of the viral cDNA sequences (FIG. 4) .
  • Recombinant VP555 encodes the putative 15 aa signal seguence preceding the N terminus of the structural protein precursor prM, the structural glycoprotein E, the nonstructural glycoprotein NSl, and the nonstructural protein NS2A (McAda et al. , 1987).
  • Recombinant VP583 encodes the putative signal sequence preceding the N terminus of E, E, NSl, and NS2A (McAda et al., 1987).
  • Recombinant vP650 contains a cDNA encoding the same proteins as vP555 with the addition of the NS2B coding region.
  • Recombinant vP658 contains a cDNA encoding the same proteins as vP583 with the addition of NS2B.
  • VP650 and vP658 a potential vaccinia virus early transcription termination signal in E (TTTTTGT; nucleotides 1087-1094) was modified to TCTTTGT without altering the aa sequence. This change was made in an attempt to increase the level of expression of E and NSl, since this sequence has been shown to increase transcription termination in in vitro transcription assays (Yuen et al., 1987).
  • FIGS. 5 and 6 show a comparison of the NSl proteins produced by JEV infection or infection with the recombinant vaccinia viruses. BHK cells were infected with
  • NSl were glycosylated. Specifically, the cell-associated forms of NSl all contained two immature (endo H sensitive) N-linked glycans, while the extracellular forms contained one immature and one complex or hybrid (endo H resistant) glycan (see Mason, 1989) . Interestingly, these pulse-chase studies showed similar levels of NSl production by all four recombinants, suggesting that the potential vaccinia early transcriptional termination signal present near the end of the E coding region in VP555 and VP583 did not significantly reduce the amount of NSl produced relative to vP650 or vP658 in which the TTTTTGT was modified. Although the experiments depicted in FIGS.
  • FIGS. 7 and 8 show a comparison of the E protein produced by JEV infection or infection with the recombinant vaccinia viruses. BHK cells were infected with JEV or recombinant vaccinia viruses, then labeled for 1 hr with
  • the extracellular fluid harvested from cells infected with vP555 and vP650 contained forms of E that migrated with a peak of hemagglutinating activity in sucrose density gradients. Interestingly, this hemagglutinin migrated similarly to the slowly sedimenting peak of noninfectious hemagglutinin (SHA) (Russell et al., 1980) found in the culture fluid of JEV-infected cells (FIG. 9) . Furthermore, these same fractions contained the fully processed form of M, demonstrating that VP555- and vP650-infected cells produced a particle that contained both of the structural membrane proteins of JEV.
  • SHA noninfectious hemagglutinin
  • the hemagglutinating properties of the supernatant fluid of cells infected with the recombinant viruses was examined, since hemagglutination activity requires particulate forms of JEV proteins that are sensitive to disruption by detergents (Eckels et al., 1975). These hemagglutination assays showed that the supernatant fluids harvested from cells infected with VP555 and VP650 contained hemagglutinating activity that was inhibited by anti-JEV antibodies and had a pH optimum identical to the JEV hemagglutinin. No hemagglutinating activity was detected in the culture fluid of cells infected with vP410, vP583, or VP658.
  • Recombinant vaccinia virus VP555 produced E- and
  • the recombinant viruses described herein contain portions cf the JEV ORF that encode the precursor to the structural protein M, the structural protein E, and nonstructural proteins NSl, NS2A, and NS2B.
  • the E and NSl proteins produced by cells infected with these recombinant viruses underwent proteolytic cleavage and N-linked carbohydrate addition in a manner indistinguishable from the same proteins produced by cells infected with JEV.
  • vP555 and vP650 differed from the remaining recombinants in that they contained the prM coding region in addition to E, NSl, and NS2A.
  • mice Groups of 3-week-old outbred Swiss mice were immunized by intraperitoneal injection with 10 7 pfu of vaccinia virus diluted in 0.1 ml of PBS. Three weeks after inoculation, selected mice were bled from the retroorbital sinus, and sera were stored at -70°C. Two to three days after bleeding, the mice were either re-inoculated with the recombinant virus or challenged by intraperitoneal injection with dilutions of suckling mouse brain infected with JEV
  • mice were observed at daily intervals for three weeks.
  • Staphylococcus aureus bacteria (The Enzyme Center, Maiden,
  • vaccinia viruses were tested for their ability to protect outbred mice from lethal JEV infection using the Beijing strain of JEV, which exhibits high peripheral pathogenicity in mice (Huang, 1982) .
  • two viruses vP555 and vP658 were selected for in-depth challenge studies.
  • vP555 induced the synthesis of extracellular forms of E
  • vP658 did not produce any extracellular forms of E, but contained additional cDNA sequences encoding the NS2B protein.
  • FIG. 10 shows an analysis of the JEV-specific reactivity of pre-cha,llenge sera from animals vaccinated with the recombinant vaccinia viruses. Sera collected from a subset of the animals used in the protection experiments (see Tables 1 and 2) were pooled and aliquots were tested for their ability to immunoprecipitate radiolabeled proteins harvested from the culture fluid of JEV-infected cells. The two lanes on the right side of the autoradiogram of FIG.
  • vP555 produces an extracellular particulate form of the structural proteins E and M.
  • This SHA-like particle probably represents an empty JEV envelope that contains E and M folded and assembled into a configuration very similar to that found in the infectious JEV particle.
  • Recombinants vP555 and vP650 may generate extracellular forms of the structural proteins because they contain the coding regions for all three JEV glycoproteins, thereby providing all of the JEV gene products needed for assembly of viral envelopes.
  • Vaccinia recombinant used for immunization Group 1 indicates animals challenged 3 weeks following a single vaccinia inoculation, and group 2 indicates animals challenged following two inoculations.
  • deletion loci were also engineered as recipient loci for the insertion of foreign genes.
  • TK thymidine kinase gene
  • HA hemagglutinin gene
  • Plasmids were constructed, screened and grown by standard procedures (Maniatis et al., 1986; Perkus et al., 1985; Piccini et al., 1987). Restriction endonucleases were obtained from GIBCO/BRL, Gaithersburg, MD, New England Biolabs, Beverly, MA; and Boehringer Mannheim Biochemicals, Indianapolis, IN. Klenow fragment of E. coli polymerase was obtained from Boehringer Mannheim Biochemicals. BAL-31 exonuclease and phage T4 DNA ligase were obtained from New England Biolabs. The reagents were used as specified by the various suppliers.
  • Synthetic oligodeoxyribonucleotides were prepared on a Biosearch 8750 or Applied Biosystems 380B DNA synthesizer as previously described (Perkus et al., 1989). DNA sequencing was performed by the dideoxy-chain termination method (Sanger et al., 1977) using Sequenase (Tabor et al. , 1987) as previously described (Guo et al., 1989) .
  • DNA amplification by polymerase chain reaction (PCR) for sequence verification was performed using custom synthesized oligonucleotide primers and GeneAmp DNA amplification Reagent Kit (Perkin Elmer Cetus, Norwalk, CT) in an automated Perkin Elmer Cetus DNA Thermal Cycler.
  • PCR polymerase chain reaction
  • GeneAmp DNA amplification Reagent Kit Perkin Elmer Cetus, Norwalk, CT
  • Excess DNA sequences were deleted from plasmids by restriction endonuclease digestion followed by limited digestion by BAL-31 exonuclease and mutagenesis (Mandecki, 1986) using synthetic oligonucleotides.
  • Cells, Virus, and Transfection The origins and conditions of cultivation of the
  • Copenhagen strain of vaccinia virus has been previously described (Guo et al., 1989) .
  • Generation of recombinant virus by recombination, in situ hybridization of nitrocellulose filters and screening for Beta-galactosidase activity are as previously described (Panicali et al., 1982; Perkus et al., 1989). Construction of Plasmid pSD 60 for Deletion of Thvmidine Kinase Gene (J2R)
  • plasmid pSD406 contains vaccinia Hindlll J (pos. 83359 - 88377) cloned into pUC8.
  • pSD406 was cut with Hindlll and PvuII, and the 1.7 kb fragment from the left side of Hindlll J cloned into pUC8 cut with Hindlll/Smal. forming pSD447.
  • pSD447 contains the entire gene for J2R (pos..83855 - 84385). The initiation codon is contained within an Nlalll site and the termination codon is contained within an Sspl site. Direction of transcription is indicated by an arrow in FIG. 11.
  • Hindlll/EcoRI fragment was isolated from pSD447, then digested with Nlalll and a 0.5 kb Hindlll/Nlalll fragment isolated. Annealed synthetic oligonucleotides
  • pSD447 was cut with Sspl (partial) within vaccinia sequences and
  • pSD460 was used as donor plasmid for recombination with wild type parental vaccinia virus Copenhagen strain VC-2.
  • 32 P labeled probe was synthesized by primer extension using MPSYN45 (SEQ ID NO:3) as template and the complementary 20mer oligonucleotide MPSYN47 (SEQ ID NO:5)
  • Recombinant virus vP410 was identified by plaque hybridization.
  • Plasmid pSD486 for Deletion of Hemorrhagic Region (B13R + B14R)
  • plasmid pSD419 contains vaccinia Sail G (pos. 160,744-173,351) cloned into pUC8.
  • pSD422 contains the contiguous vaccinia Sail fragment to the right, Sail J (pos. 173,351-182,746) cloned into pUC8.
  • Sail J pos. 173,351-182,746
  • B13R - B14R (pos. 172,549 - 173,552)
  • pSD419 was used as the source for the left flanking arm
  • pSD422 was used as the source of the right flanking arm.
  • the direction of transcription for the u region is indicated by an arrow in FIG. 12.
  • sequences to the left of the Ncol site were removed by digestion of pSD419 with Ncol/Smal followed by blunt ending with Klenow fragment of E. coli polymerase and ligation generating plasmid pSD476.
  • a vaccinia right flanking arm was obtained by digestion of pSD422 with Hpal at the termination codon of B14R and by digestion with Nrul 0.3 kb to the right. This 0.3 kb fragment was isolated and ligated with a 3.4 kb Hindi vector fragment isolated from pSD476, generating plasmid pSD477.
  • the location of the partial deletion of the vaccinia u region in pSD477 is indicated by a triangle.
  • the remaining B13R coding sequences in pSD477 were removed by digestion with Clal/Hpal, and the resulting vector fragment was ligated with annealed synthetic oligonucleotides SD22mer/SD20mer
  • pSD479 contains an initiation codon (underlined) followed by a BamHI site.
  • E. coli Beta-galactosidase in the B13-B14 (u) deletion locus under the control of the u promoter, a 3.2 kb BamHI fragment containing the Beta-galactosidase gene (Shapira et al., 1983) was inserted into the BamHI site of pSD479, generating pSD479BG.
  • pSD479BG was used as donor plasmid for recombination with vaccinia virus vP410.
  • Recombinant vaccinia virus vP533 was isolated as a blue plaque in the presence of chromogenic substrate X-gal.
  • vP533 the B13R- B14R region is deleted and is replaced by Beta- galactosidase.
  • plasmid pSD486 a derivative of pSD477 containing a polylinker region but no initiation codon at the u deletion junction, was utilized.
  • Clal/Hpal vector fragment from pSD477 referred to above was ligated with annealed synthetic oligonucleotides SD42mer/SD40mer (SEQ ID NO:8/SEQ ID NO:9)
  • pSD486 was used as donor plasmid for recombination with recombinant vaccinia virus vP533, generating vP553, which was isolated as a clear plaque in the presence of X-gal. Construction of Plasmid pMP494A for Deletion of ATI Region (A26L)
  • pSD414 contains Sail B cloned into pUC8.
  • pSD414 was cut with Xbal within vaccinia sequences (pos. 137,079) and with Hindlll at the pUC/vaccinia junction, then blunt ended with Klenow fragment of E. coli polymerase and ligated, resulting in plasmid pSD483.
  • pSD483 was cut with EcoRI (pos. 140,665 and at the pUC/vaccinia junction) and ligated, forming plasmid pSD484.
  • pSD484 was cut with Ndel (partial) slightly upstream from the A26L ORF (pos. 139,004) and with Hpal (pos. 137,889) slightly downstream from the A26L ORF.
  • the 5.2 kb vector fragment was isolated and ligated with annealed synthetic oligonucleotides ATI3/ATI4 (SEQ ID NO:12/SEQ ID NO:13)
  • Beta-galactosidase gene (Shapira et al., 1983) under the control of the vaccinia 11 kDa promoter (Bertholet et al., 1985; Perkus et al., 1990) was inserted into the Bqlll site of pSD492, forming pSD493KBG. Plasmid pSD493KBG was used in recombination with rescuing virus vP553. Recombinant vaccinia virus, VP581, containing Beta-galactosidase in the A26L deletion region, was isolated as a blue plaque in the presence of X-gal.
  • vaccinia DNA encompassing positions [137,889 - 138,937], including the entire A26L ORF is deleted.
  • vaccinia Sail G restriction fragment (pos. 160,744-173,351) crosses the Hindlll A/B junction (pos. 162,539).
  • pSD419 contains vaccinia Sail G cloned into pUC8.
  • the direction of transcription for the hemagglutinin (HA) gene is indicated by an arrow in FIG. 14.
  • Vaccinia sequences derived from Hindlll B were removed by digestion of pSD419 with Hindlll within vaccinia sequences and at the pUC/vaccinia junction followed by ligation.
  • the resulting plasmid, pSD456, contains the HA gene, A56R, flanked by 0.4 kb of vaccinia sequences to the left and 0.4 kb of vaccinia sequences to _ 3 _ .
  • Beta-galactosidase sequences were deleted from vP708 using donor plasmid pSD467.
  • pSD467 is identical to pSD466, except that EcoRI, Smal and BamHI sites were removed from the pUC/vaccinia junction by digestion of pSD466 with EcoRI/BamHI followed by blunt ending with Klenow fragment of E . coli polymerase and ligation.
  • Recombination between vP708 and pSD467 resulted in recombinant vaccinia deletion mutant, VP723, which was isolated as a clear plaque in the presence of X-gal.
  • pSD420 is Sail H cloned into pUC8.
  • pSD435 is Kpnl F cloned into pUC18.
  • pSD435 was cut with SphI and religated, forming pSD451.
  • DNA sequences to the left of the SphI site (pos. 27,416) in Hindlll M are removed (Perkus et al.,
  • pSD409 is Hindlll M cloned into pUC8.
  • [C7L-K1L] gene cluster from vaccinia, E. coli Beta- galactosidase was first inserted into the vaccinia M2L deletion locus (Guo et al., 1990) as follows. To eliminate the Bqlll site in pSD409, the plasmid was cut with Bqlll in vaccinia sequences (pos. 28,212) and with BamHI at the pUC/vaccinia junction, then ligated to form plasmid pMP409B. pMP409B was cut at the unique SphI site (pos. 27,416). M2L coding sequences were removed by mutagenesis (Guo et al.,
  • the resulting plasmid, pMP409D contains a unique Bglll site inserted into the M2L deletion locus as indicated above.
  • a 3.2 kb BamHI (partial) /Bqlll cassette containing the E. coli Beta-galactosidase gene (Shapira et al., 1983) under the control of the 11 kDa promoter (Bertholet et al., 1985) was inserted into pMP409D cut with Bqlll.
  • the resulting plasmid, pMP409DBG (Guo et al., 1990), was used as donor plasmid for recombination with rescuing vaccinia virus vP723.
  • Recombinant vaccinia virus, vP784, containing Beta- galactosidase inserted into the M2L deletion locus was isolated as a blue plaque in the presence of X-gal.
  • a plasmid deleted for vaccinia genes [C7L-K1L] was assembled in pUC8 cut with Smal. Hindlll and blunt ended with Klenow fragment of E. coli polymerase.
  • the left flanking arm consisting of vaccinia Hindlll C sequences was obtained by digestion of pSD420 with Xbal (pos. 18,628) followed by blunt ending with Klenow fragment of E. coli polymerase and digestion with Bqlll (pos. 19,706).
  • the right flanking arm consisting of vaccinia Hindlll K sequences was obtained by digestion of pSD451 with Bglll (pos. 29,062) and EcoRV (pos. 29,778).
  • the resulting plasmid, pMP581CK is deleted for vaccinia sequences between the Bqlll site (pos. 19,706) in Hindlll C and the Bqlll site (pos. 29,062) in Hindlll K.
  • the site of the deletion of vaccinia sequences in plasmid pMP581CK is indicated by a triangle in FIG. 15.
  • plasmid pSD405 contains vaccinia Hindlll I (pos. 63,875-70,367) cloned in pUC8. pSD405 was digested with EcoRV within vaccinia sequences
  • pSD518 was used as the source of all the vaccinia restriction fragments used in the construction of pSD548.
  • the vaccinia I4L gene extends from position
  • pSD518 was digested with BamHI (pos. 65,381) and Hpal (pos. 67,001) and blunt ended using Klenow fragment of E . coli polymerase.
  • This 4.8 kb vector fragment was ligated with a 3.2 kb Smal cassette containing the E. coli Beta- galactosidase gene (Shapira et al. , 1983) under the control of the vaccinia 11 kDa promoter (Bertholet et al., 1985; Perkus et al., 1990), resulting in plasmid pSD524KBG.
  • pSD524KBG was used as donor plasmid for recombination with vaccinia virus vP804.
  • deletion plasmid pSD548 was constructed.
  • the left and right vaccinia flanking arms were assembled separately in pUC8 as detailed below and presented schematically in FIG. 16.
  • pUC8 was cut with BamHI/EcoRI and ligated with annealed synthetic oligonucleotides 518A1/518A2
  • pSD531 was cut with Rsal (partial) and BamHI and a 2.7 kb vector fragment isolated.
  • pSD518 was cut with Bqlll (pos. 64,459)/ Rsal (pos. 64,994) and a 0.5 kb fragment isolated. The two fragments were ligated together, forming pSD537, which contains the complete vaccinia flanking arm left of the I4L coding sequences.
  • pUC8 was cut with BamHI/EcoRI and ligated with annealed synthetic oligonucleotides 518B1/518B2 (SEQ ID NO:23/SEQ ID NO:24) ., ⁇
  • pSD532 was cut with Rsal
  • the vaccinia I4L deletion cassette was moved from pSD539 into pRCll, a pUC derivative from which all Beta- galactosidase sequences have been removed and replaced with a polylinker region (Colinas et al., 1990).
  • pSD539 was cut with EcoRI/PstI and the 1.2 kb fragment isolated. This fragment was ligated into pRCll cut with EcoRI/PstI (2.35 kb) , forming pSD548.
  • GLYCOPROTEINS cDNA copies of the sequences encoding the HA and F proteins of measles virus MV (Edmonston strain) were inserted into NYVAC to create a double recombinant designated NYVAC-MV.
  • the recombinant authentically expressed both measles glycoproteins on the surface of infected cells. Immunoprecipitation analysis demonstrated correct processing of both F and HA glycoproteins. The recombinant was also shown to induce syncytia formation.
  • NYVAC-MV was the modified Copenhagen strain of vaccinia virus designated NYVAC. All viruses were grown and titered on Vero cell monolayers. Plasmid Construction
  • Plasmid pSPM2LHA (Taylor et al. , 1991) contains the entire measles HA gene linked in a precise ATG to ATG configuration with the vaccinia virus H6 promoter which has been previously described (Taylor et al., 1988a,b; Guo et al., 1989; Perkus et al., 1989).
  • a 1.8kpb EcoRV/Smal fragment containing the 3• most 24 bp of the H6 promoter fused in a precise ATG:ATG configuration with the HA gene lacking the 3' most 26 bp was isolated from pSPM2LHA. This fragment was used to replace the 1.8 kbp EcoRV/Smal fragment of pSPMHHAll (Taylor et al., 1991) to generate pRW803.
  • Plasmid pRW803 contains the entire H6 promoter linked precisely to the entire measles HA gene.
  • sequence for codon 18(CCC) was deleted as compared to the published sequence (Alkhatib et al., 1986).
  • the CCC sequence was replaced by oligonucleotide mutagenesis via the Kunkel method (Kunkel, 1985) using oligonucleotide RW117 (SEQ ID NO:39) (5'GACTATCCTACTTCCCTTGGGATGGGGGTTATCTTTGTA-3•) .
  • PRO 18 Single stranded template was derived from plasmid pRW819 which contains the H6/HA cassette from pRW803 in pIBI25
  • the mutagenized plasmid containing the inserted (CCC) to encode for a proline residue at codon 18 was designated pRW820.
  • the Hindlll site is situated at the 5' border of the H6 promoter while the Xbal site is located 230 bp downstream from the initiation codon of the HA gene.
  • the mutagenized expression cassette contained within pRW837 was derived by digestion with Hindlll and EcoRI, blunt-ended using the Klenow fragment of E. coli DNA polymerase in the presence of 2mM dNTPs, and inserted into the Smal site of pSD513 to yield pRW843.
  • Plasmid pSD513 was derived from plasmid pSD460 by the addition of polylinker sequences. Plasmid pSD460 was derived to enable deletion of the thymidine kinase gene from vaccinia virus (FIG. 11) .
  • the resultant plasmid was designated pSPMF75M20.
  • the plasmid pSPMF75M20 which contains the measles
  • F gene now linked in a precise ATG for ATG configuration with the H6 promoter was digested with Nrul and EaqI.
  • the resulting 1.7 kbp blunt ended fragment containing the 3 • most 27 bp of the H6 promoter and the entire fusion gene was isolated and inserted into an intermediate plasmid pRW823 which had been digested with Nrul and Xbal and blunt ended.
  • the resultant plasmid pRW841 contains the H6 promoter linked to the measles F gene in the pIBI25 plasmid vector (International Biotechnologies, Inc., New Haven, CT) .
  • the H6/measles F cassette was excised from pRW841 by digestion with Smal and the resulting 1.8 kb fragment was inserted into pRW843 (containing the measles HA gene) .
  • Plasmid pRW843 was first digested with NotI and blunt-ended with Klenow fragment of E. coli DNA polymerase in the presence of 2mM dNTPs.
  • the resulting plasmid, pRW857 therefore contains the measles virus F and HA genes linked in a tail to tail configuration. Both genes are linked to the vaccinia virus H6 promoter.
  • Development of NYVAC-MV Plasmid pRW857 was transfected into NYVAC infected
  • Vero cells by using the calcium phosphate precipitation method previously described (Panicali et al., 1982; Piccini et al., 1987). Positive plaques were selected on the basis of in situ plaque hybridization to specific MV F and HA radiolabeled probes and subjected to 6 sequential rounds of plaque purification until a pure population was achieved.
  • a thymidine kinase mutant of the Copenhagen strain of vaccinia virus vP410 (Guo et al. , 1989) was used to generate recombinants vP825, VP829, vP857 and VP864 (see below) .
  • the generation of vP555 has previously been described (Mason et al., 1991). All vaccinia virus stocks were produced in VERO (ATCC CCL81) cells in Eagle's minimal essential medium plus 10% heat inactivated fetal bovine serum (FBS) .
  • FBS heat inactivated fetal bovine serum
  • cDNA encoding the C protein of JEV was obtained by a modification of the method of Okayama and Berg (1982) using Moloney murine leukemia virus reverse transcriptase (GIBCO/BRL, Gaithersburg, MD) (D'Alessio and Gerrard, 1988).
  • Genomic RNA was isolated from virions prepared by the method of Repik et al. (1983) from suspension cultures of C6/36 cells infected with a passage 55 suckling mouse brain stock of the Nakayama strain of JEV.
  • First strand cDNA synthesis was primed from a synthetic oligonucleotide complementary to bases 986 to 1005 of the E coding region of JEV (FIG. 17A and B) (SEQ ID NO:52).
  • the double-stranded cDNA was ligated to synthetic oligonucleotides containing the EcoRI site (New England Biolabs, Beverly, MA) , inserted into phosphatase treated EcoRI-cleaved pBR322 (New England Biolabs) , and the resulting DNA was used to transform E. coli strain DH5 cells (GIBCO/BRL) . Plasmids were analyzed by restriction enzyme digestion and a plasmid (pC20) containing cDNA corresponding to 81 nucleotides of non-coding RNA and the C and prM coding regions was identified.
  • pC20 plasmid
  • pC20 was digested at the linker sites with EcoRI and at an internal Dral site situated 28 bp 5• of the ATG initiation codon and the resulting fragment containing the C and prM coding regions was inserted into Smal-EcoRI digested pUC18, creating plasmid, pDr20.
  • the sequence of the C coding region of pC20, combined with an updated sequence of the prM, E, NSl, NS2A, and NS2B coding regions of the Nakayama strain of JEV is presented in FIG. 17A and B (SEQ ID NO:52). All nucleotide coordinates are based on this updated sequence with numbering beginning at the C protein Met initiation codon.
  • TTTTTGT nucleotides 1304 to 1310 were changed to TCTTTGT
  • NS2B nucleotides 2124 to 4126
  • the plasmid origin and vaccinia sequences was ligated to the purified Smal-SacI insert from JEV20 yielding JEV22-1.
  • the 6 bp corresponding to the unique Smal site used to construct JEV22-1 were removed using oligonucleotide-directed double-strand break mutagenesis (Mandecki, 1986) creating JEV24 in which the H6 promoter immediately preceded the ATG start codon.
  • Plasmid JEV7 (FIG. 2) was digested with SphI within JE sequences (nucleotide 2381) and Hindlll within IBI24. Ligation to annealed oligonucleotides J94 and J95 [containing a SphI sticky end, translation stop, a vaccinia early transcription termination signal (TTTTTAT; Yuen et al., 1987) a translation stop, an EaqI site and a Hindlll sticky end] generated plasmid JEV25 which contains JE cDNA extending from the SacI site (nucleotide 2124) in the last third of E through the carboxy-terminus of E.
  • TTTTTAT vaccinia early transcription termination signal
  • SacI-EagI fragment from JEV25 was ligated to the SacI-EagI fragment of JEV8 (containing JE cDNA encoding 15 aa C, prM and amino- terminal two thirds of E nucleotides 337 to 2124, the plasmid origin and vaccinia sequences) yielding plasmid JEV26.
  • a unique Smal site preceding the ATG start codon was removed as described above, creating JEV27 in which the H6 promoter immediately preceded the ATG start codon.
  • Oligonucleotides J96, J97, J98 and J99 (containing
  • JEV28 was digested with Hpal within the JE sequence (nucleotide 2301) and with Hindlll within the pIBI25 sequence and alkaline phosphatase treated. Ligation to the Hpal-Hindlll fragment from JEV1 or Hpal-Hindlll fragment from JEV7 (FIG.
  • JEV29 [containing a Smal site followed by JE cDNA encoding 30 aa E, NSl, NS2A (nucleotides 2293 to 4125)
  • JEV30 [containing a Smal site followed by JE cDNA encoding 30 aa E, NSl, NS2A, NS2B (nucleotides 2293 to 4512) ] .
  • the Smal-EagI fragment from JEV29 was ligated to Smal-EagI digested pTP15 (Mason et al., 1991) yielding JEV31.
  • the 6 bp corresponding to the unique Smal site used to produce JEV31 were removed as described above creating JEV33 in which the H6 promoter immediately preceded the ATG start codon.
  • the Smal-EagI fragment from JEV30 was ligated to Smal-EagI digested pTP15 yielding JEV32.
  • the 6 bp corresponding to the unique Smal site used to produce JEV32 were removed as described above creating JEV34 in which the H6 promoter immediately preceded the ATG start codon.
  • HeLa cell monolayers were prepared in 35 mm diameter dishes and infected with vaccinia viruses (m.o.i. of 2) or JEV (m.o.i. of 5) before radiolabeling.
  • vaccinia viruses m.o.i. of 2
  • JEV m.o.i. of 5
  • cells were pulse labeled with medium containing 35 S-Met and chased for 6 hr in the presence of excess unlabeled Met exactly as described by Mason et al. (1991) .
  • JEV-infected cells were radiolabeled as above for preparation of radioactive proteins for checking pre- and post-challenge mouse sera by radioimmunoprecipitation. adioimmunoprecipitations, Polv cryl ⁇ wi-a-a flei
  • Radiolabeled cell lysates and culture fluids were harvested and the viral proteins were immunoprecipitated, digested with endoglycosidases, and separated in SDS- containing polyacrylamide gels (SDS-PAGE) exactly as described by Mason (1989) .
  • mice were immunized by intraperitoneal (ip) injection with 10 7 pfu of vaccinia virus, and 3 weeks later sera were collected from selected mice. Mice were then either re-inoculated with the recombinant virus or challenged by ip injection with a suspension of suckling mouse brain infected with the P3 strain of JEV. Three weeks later, the boosted animals were re-bled and challenged with the P3 strain of JEV. Following challenge, mice were observed at daily intervals for three weeks and lethal-dose titrations were performed in each challenge experiment using litter-mates of the experimental animals. In addition, sera were collected from all surviving animals 4 weeks after challenge.
  • vaccinia recombinants in the HA locus were constructed that expressed portions of the JEV coding region extending from C through NS2B.
  • the JEV cDNA sequences contained in these recombinant viruses are shown in FIG. 18.
  • the sense strand of the JEV cDNA was positioned behind the vaccinia virus early/late H6 promoter, and translation was expected to be initiated from naturally occurring JEV Met codons located at the 5' ends of the viral cDNA sequences.
  • Recombinant VP825 encoded the capsid protein C, structural protein precursor prM, the structural glycoprotein E, the nonstructural glycoprotein NSl, and the nonstructural protein NS2A (McAda et al., 1987).
  • Recombinant vP829 encoded the putative 15 aa signal sequence preceding the amino-terminus of prM, as well as prM, and E (McAda et al., 1987).
  • Recombinant VP857 contained a cDNA encoding the 30 aa hydrophobic carboxy-terminus of E, followed by NSl and NS2A.
  • Recombinant vP864 contained a cDNA encoding the same proteins as vP857 with the addition of NS2B.
  • vaccinia virus early transcription termination signal in E was modified to TCTTTGT without altering the aa sequence. This change was made in an attempt to increase the level of expression of E since this sequence has been shown to increase transcription termination in in vitro transcription assays (Yuen et al.,
  • Pulse-chase experiments demonstrate that proteins identical in size to E were synthesized in cells infected with all recombinant vaccinia viruses containing the E gene (Table 3) .
  • an E protein that migrated slower in SDS-PAGE was also detected in the culture fluid harvested from the infected cells (Table 3) .
  • This extracellular form of E produced by JEV- and vP555-infected cells contained mature N-linked glycans (Mason, 1989; Mason et al., 1991), as confirmed for the extracellular forms of E produced by vP829-infected cells.
  • VP825 which contained the C coding region in addition to prM and E specified the synthesis of E in a form that is not released into the extracellular fluid (Table 3) .
  • the extracellular fluid harvested from cells infected with VP555 and vP829 contained an HA activity that was not detected in the culture fluid of cells infected with vP410, VP825, vP857 or vP864.
  • the HA activity observed in the culture fluid of vP829 infected cells was 8 times as high as that obtained from vP555 infected cells. This HA appeared similar to the HA produced in JEV infected cells based on its inhibition by anti-JEV antibodies and its pH optimum (Mason et al., 1991).
  • sucrose density gradients prepared with culture fluids obtained from infected cells identified a peak of HA activity in the vP829 sample that co-migrated with the peak of slowly sedimented hemagglutinin (SHA) found in the JEV culture fluids (Table
  • NSl and NSl' were synthesized in cells infected with vP555, VP825, VP857 and VP864 (Table 3).
  • NSl produced by VP555- infected cells was released into the culture fluid of infected cells in a higher molecular weight form.
  • NSl was also released into the culture fluid of cells infected with vP857 and VP864 (Table 3).
  • the efficiency of release of NSl by vP825 infected cells was more than 10 times less than that for NSl synthesized in vP555, vP857 or vP864 infected cells.
  • Plasmid pMP2VCL (containing a polylinker region within vaccinia sequences upstream of the K1L host range gene) was digested within the polylinker with Hindlll and
  • Plasmid pSD544VC (containing vaccinia sequences surrounding the site of the HA gene replaced with a polylinker region and translation termination codons in six reading frames) was digested with Xhol within the polylinker, filled in with the Klenow fragment of DNA polymerase I and treated with alkaline phosphatase. SP126 was digested with Hindlll, treated with Klenow and the H6 promoter isolated by digestion with Smal. Ligation of the H6 promoter fragment to pSD544VC generated SPHA-H6 which contained the H6 promoter in the polylinker region (in the direction of HA transcription) .
  • Plasmid JEVL14VC (FIG. 1) was digested with EcoRV in the H6 promoter and SacI in JEV sequences (nucleotide 2124) and a 1789 bp fragment isolated. JEVL14VC was digested with EclXI at the EaqI site following the T5NT, filled in with the Klenow fragment of DNA polymerase I and digested with SacI in JEV sequences (nucleotide 2124) — i—
  • JEV35 was digested with SacI (within JE sequences nucleotide 2124) and EclXI (after T5NT) a 5497 bp fragment isolated and ligated to a SacI (JEV nucleotide 2125) to EaqI fragment of JEV25 (containing the remaining two thirds of E, translation stop and T5NT) generating JEV36.
  • JEV36 was transfected into vP866 (NYVAC) infected cells to generate the vaccinia recombinant VP923 (FIG. 18) .
  • Oligonucleotides SPHPRHA A through D (SEQ ID NO:31), (SEQ ID NO:32), (SEQ ID NO:33) and (SEQ ID NO:34) are ligated to generate the following sequences (SEQ ID NO:56/SEQ ID NO:57)
  • mice were immunized by intraperitoneal (ip) injection of 10 7 pfu of vaccinia virus, and 3 weeks later sera were collected from selected mice. Mice were then challenged by ip injection with a suspension of suckling mouse brain infected with the P3 strain of JEV (multiple mouse passage; Huang, 1982) . Following challenge mice were observed daily for three weeks. Evaluation ⁇ > ⁇ Tmmiitie Res onse to JEV NYVAC P «»r * !Q-mfrH ⁇ »a ⁇ fc9
  • HAI tests were performed as described by Mason et al. (1991) .
  • NYVAC recombinants vP908 and VP923 elicited high levels of hemagglutination-inhibiting antibodies and protected mice against more than 100,000 LD 50 of JEV (Table
  • NYVAC (VP866) ⁇ 1:10 0/12 VP908 1:80 11/12
  • a host range mutant of vaccinia virus (WR strain) vP293 (Perkus et al. , 1989) was used to generate all recombinants (see below) .
  • All vaccinia virus stocks were produced in either VERO (ATCC CCL81) or MRC-5 (ATCC CCL171) cells in Eagles MEM supplemented with 5-10% newborn calf serum (Flow Laboratories, McLean, VA) .
  • the YF 17D cDNA clones used to construct the YF vaccinia recombinant viruses were obtained from Charles Rice (Washington University
  • nucleotide coordinates are derived from the sequence data presented in
  • Plasmid YF0 containing YF cDNA encoding the carboxy-terminal 80% prM, E and amino-terminal 80% NSl was derived by cloning an Aval to Nsil fragment of YF cDNA (nucleotides 537-1658) and an Nsil to Kpnl fragment of YF cDNA (nucleotides 1659-3266) into Aval and Kpnl digested IBI25 (International Biotechnologies, Inc. , New Haven, CT) .
  • Plasmid YF1 containing YF cDNA encoding C and amino-terminal 20% prM was derived by cloning a Rsal to Aval fragment of YF cDNA (nucleotides 166-536) and annealed oligos SP46 and SP47 (containing a disabled Hindlll sticky end, Xhol and Clal sites and YF nucleotides 119-165) into Aval and Hindlll digested IBI25.
  • Plasmid YF3 containing YF cDNA encoding the carboxy-terminal 60% of E and amino-terminal 25% of NSl was generated by cloning an Apal to BamHI fragment of YF cDNA (nucleotides 1604-2725) into Apal and BamHI digested IBI25.
  • Plasmid YF8 containing YF cDNA encoding the carboxy-terminal 20% NSl NS2A, NS2B and amino-terminal 20% NS3 was derived by cloning a Kpnl to Xbal fragment of YF cDNA (nucleotides 3267-4940) into Kpnl and Xbal digested IBI25.
  • Plasmid YF9 containing YF cDNA encoding the carboxy-terminal 60% NS2B and amino-terminal 20% NS3 was generated by cloning a SacI to Xbal fragment of YF cDNA (nucleotides 4339-4940) into Sa and Xbal digested IBI25.
  • Plasmid YF13 containing YF cDNA encoding the carboxy-terminal 25% of C, prM and amino- terminal 40% of E was derived by cloning a Ball to Apal fragment of YF cDNA (nucleotides 384-1603) into Apal and Smal digested IBI25.
  • Oligonucleotide-directed mutagenesis was used to change potential vaccinia virus early transcription termination signals (Yuen et al., 1987) 49 aa from the amino-terminus of the C gene in YF1 (TTTTTCT nucleotides 263-269 and TTTTTGT nucleotides 269-275) to (SEQ ID NO:35) TTCTTCTTCTTGT creating plasmid YF1B, in the E gene in YF3 (nucleotides 1886-1893 TTTTGT to TTCTTTGT 189 aa from the carboxy-terminus and nucleotides 2429-2435 TTTTTGT to TTCTTGT 8 aa from the carboxy-terminus) creating plasmids YF3B and YF3C.
  • Plasmid YF6 was digested with EcoRV within the IBI25 sequences and Aval at nucleotide 537 and ligated to an EcoRV to Aval fragment from YF1B (EcoRV within IBI25 to Aval at nucleotide 536) generating YF2 containing YF cDNA encoding C through the amino-terminal 80% of NSl (nucleotides 119-3266) with an Xhol and Clal site at 119 and four mutagenized transcription termination signals.
  • Oligonucleotide-directed mutagenesis described above was used to insert Xhol and Clal sites preceding the ATG 17 aa from the carboxy-terminus of E (nucleotides 2402- 2404) in plasmid YF3C creating YF5, to insert Xhol and Clal sites preceding the ATG 19 aa from the carboxy-terminus of prM (nucleotides 917-919) in plasmid YF13 creating YF14, to insert an Xhol site preceding the ATG 23 aa from the carboxy-terminus of E (nucleotides 2384-2386) in plasmid YF3C creating plasmid YF25, and to insert an Xhol site and ATG (nucleotide 419) in plasmid YF1 21 aa from the carboxy- terminus of C generating YF45.
  • nucleotides 1604-2725 was exchanged for the corresponding region of YFO generating YF26 containing YF cDNA encoding the carboxy-terminal 80% prM, E and amino-terminal 80% NSl (nucleotides 537-3266) with an Xhol site at nucleotide 2384 (23 aa from the carboxy-terminus of E) and mutagenized transcription termination signal at 2428-2435 (8 aa from the carboxy-terminus of E) .
  • YF6 was digested within IBI25 with EcoRV and within YF at nucleotide 537 with Aval and ligated to EcoRV (within IBI25) to Aval fragment of YF45 generating YF46 containing YF cDNA encoding C through the amino-terminal 80% NSl (nucleotides 119-3266) with an Xhol site at 419 (21 aa from the carboxy-terminus of C) and two transcription termination signals removed.
  • Oligonucleotide-directed mutagenesis described above was used to insert a Smal site at the carboxy-terminus of NS2B (nucleotide 4569) in plasmid YF9 creating YF11, and to insert a Smal site at the carboxy-terminus of NS2A (nucleotide 4180) in plasmid YF8 creating YF10.
  • Plasmid pHES4 contains the vaccinia K1L host range gene, the early/late vaccinia virus H6 promoter, unique multicloning restriction sites, translation stop codons and an early transcription termination signal (Perkus et al., 1989) .
  • a Kpnl to Smal fragment from YF12 encoding carboxy- terminal 20% NSl, NS2A and NS2B (nucleotides 3267-4569) , Xhol to Kpnl fragment from YF15 encoding 19 aa prM, E and amino-terminal 80% NSl (nucleotides 917-3266) and Xh -Smal digested pHES4 were ligated generating YF23.
  • Xhol-Smal digested pHES4 was ligated to a purified Xhol to Kpnl fragment from YF7 encoding 17 aa E and amino- terminal 80% NSl (nucleotides 2402-3266) plus a Kpnl to Smal fragment from YF10 encoding the carboxy-terminal 20% NSl and NS2A (nucleotides 3267-4180) creating YF18.
  • vP457 containing a host range gene restored in the vP293 background has been described (Perkus et al., 1989).
  • MEM Met-free media
  • cells were pulsed labeled with medium containing 35 S-Met and chased for 6 hr in the presence of excess unlabeled Met.
  • Radiolabeled cell lysates and culture fluids were harvested and the viral proteins were immunoprecipitated with monoclonal antibodies to YF E and NSl and separated in SDS-containing polyacrylamide gels exactly as described by Mason (1989) .
  • Animal Protection Experiments Groups of 3 week old mice were immunized by intraperitoneal injection with 10 7 pfu of vaccinia virus or 100 ⁇ l of a 10% suspension of suckling mouse brain containing YF17D. Three weeks later sera were collected from selected mice. Mice were then either re-inoculated with the recombinant virus or YF17D, or challenged by i.c. injection of the French Neurotropic strain of YFV.
  • vaccinia virus recombinants Five different vaccinia virus recombinants that expressed portions of the YF coding region extending from C through NS2B were constructed utilizing a host range selection system (Perkus et al., 1989). The YF cDNA sequences contained in these recombinants are shown in FIG. 19. In all five recombinant viruses the sense strand of YF cDNA was positioned behind the vaccinia virus early/late H6 promoter, and translation was expected to be initiated from Met codons located at the 5' ends of the viral cDNA sequences (FIG. 19) .
  • Recombinant vP725 encoded the putative 17-aa signal sequence preceding the N terminus of the nonstructural protein NSl and the nonstructural proteins NSl and NS2A (Rice et al., 1985).
  • Recombinant VP729 encoded the putative 19-aa signal sequence preceding the N terminus of E, E, NSl, NS2A and NS2B (Rice et al., 1985).
  • Recombinant vP764 encoded C, prM, E, NSl, NS2A and NS2B (Rice et al. , 1985) .
  • the extracellular fluid harvested from cells infected with VP869 contained an HA activity that was not detected in the culture fluid of VP766, VP729, VP725, or vP457 infected cells (Table 7) .
  • This HA appeared similar to the HA produced in YF17D infected cells based on its pH optimum.
  • NSl Protein Expression by Recombinant Vaccinia Virus The results of pulse-chase experiments in HeLa cells demonstrated that proteins identical in size to authentic YF17D NSl were synthesized in cells infected with VP725, vP766, and VP729 (Table 1) , however, the amounts synthesized greatly varied.
  • NSl produced by VP725 and VP729 infected cells was released into the culture fluid of infected cells in a higher molecular weight form similar to NSl secreted by YF17D infected cells.
  • vP766 infected cells did not secrete NSl, however, the level of intracellular NSl was lowest with this recombinant (Table 7) .
  • the failure of vP869 to synthesize NSl is due to the deletion of a base
  • nucleotide 2962 in the donor plasmid (YF47) used to generate this recombinant. Protection From Lethal YF Challenge
  • vP869 In an initial experiment VP457, vP764, and vP869 were compared with YF17D in their ability to protect mice from a lethal challenge with the French Neurotropic strain of YFV (Table 8, Experiment I). vP869 provided significant protection whereas vP764 offered no better protection than the control vaccinia virus VP457.
  • Pre-challenge sera pooled from selected animals in each group were tested for their ability to immunoprecipitate radiolabeled E and NSl proteins and for the presence of Neut and HAI antibodies.
  • Table 9 only vP869 and YF17D immunized mice responded to E protein, the response was increased by a second inoculation. Mice immunized twice with vP729, vP725 or vP766 produced antibody to NSl.
  • mice were inoculated ip with 10 7 pfu vaccinia recombinant or lOO ⁇ l of a 10% suspension of suckling mouse brain containing YF17D and challenged three weeks later ic with 220 LD 50 French Neurotropic strain YFV.
  • mice were inoculated twice three weeks apart ip with 10 7 pfu vaccinia recombinant or lOO ⁇ l of a 10% suspension of suckling mouse brain containing YF17D and challenged three weeks later ic with 36 LD 50 French Neurotropic strain YFV.
  • Group I indicates animals challenged three weeks following a single inoculation.
  • Group II indicates animals challenged following two inoculations.
  • Group I indicates animals challenged three weeks following a single inoculation.
  • Group II indicates animals challenged following two inoculations.
  • a Xhol to Smal fragment from YF47 (nucleotides 419-4180) containing YF cDNA encoding 21 amino acids C, prM, E, NSl, NS2A (with a base missing in NSl nucleotide 2962) was ligated to Xhol-Smal digested SPHA-H6 (HA region donor plasmid) generating YF48.
  • YF48 was digested with Sad (nucleotide 2490) and partially digested with Asp7l8 (nucleotide 3262) and a 6700 bp fragment isolated (containing the plasmid origin of replication, vaccinia sequences, 21 amino acids C, prM, E, amino-terminal 3.5% NSl, carboxy-terminal 23% NSl, NS2A) and ligated to a Sacl- Asp7l8 fragment from YF18 (containing the remainder of NSl with the base at 2962) generating YF51.
  • YF50 encoding YF 21 amino acids C, prM, E, NSl, NS2A in the HA locus donor plasmid.
  • YF50 was transfected into vP866 (NYVAC) infected cells generating the recombinant vP984 (FIG. 19) .
  • YF50 was transfected into VP913 infected cells (NYVAC-MV) generating the recombinant VP1002 (FIG. 19) .
  • An Apal-Smal fragment of YF49 (containing the plasmid origin of replication, vaccinia sequences and YF cDNA encoding 21 amino acids C, prM, and amino-terminal 43% E) was ligated to an Apal-Smal fragment from YF16 (nucleotides 1604-2452 containing the carboxy- terminal 57% E) generating YF53 containing 21 amino acids C, prM, E in the HA locus donor plasmid.
  • YF53 was transfected into vP866 (NYVAC) infected cells generating the recombinant VP1003 (FIG. 19) .
  • YF53 was transfected into VP913 infected cells (NYVAC-MV) generating the recombinant vP997 (FIG. 19) .
  • the DEN cDNAs used to construct the DEN vaccinia recombinants were derived from a Western Pacific strain of DEN-1 (Mason et al., 1987b). Nucleotide coordinates 1-3745 are presented in that publication.
  • FIG. 20 presents the sequence of nucleotides 3392 to 6117.
  • Plasmid DENl containing DEN cDNA encoding the carboxy-terminal 84% NSl and amino-terminal 45% NS2A was derived by cloning an EcoRI-Xbal fragment of DEN cDNA (nucleotides 2579-3740) and annealed oligonucleotides DENl (SEQ ID NO:38) and DEN2 (SEQ ID NO:39) (containing a Xbal sticky end, translation termination codon, T5AT vaccinia virus early transcription termination signal Yuen et al. (1987) , EaqI site and Hindlll sticky end) into Hindlll-EcoRI digested pUC8.
  • An EcoRI-Hindlll fragment from DENl (nucleotides 2559-3745, Mason et al., 1987B) was derived by cloning an EcoRI-Xbal fragment of DEN cDNA (nucleotides 2579-3740) and annealed
  • Hindlll-Sacl digested IBI24 International Biotechnologies, Inc., New Haven, CT
  • DEN3 encoding the carboxy- terminal 64% E through amino-terminal 45% NS2A with a base missing in NSl (nucleotide 2467) .
  • Hindlll-Xbal digested IBI24 was ligated to annealed oligonucleotides DEN9 (SEQ ID NO:40) and DEN10 (SEQ ID NO:41) [containing a Hindlll sticky end, Smal site, DEN nucleotides 377-428 (Mason et al., 1987B) and Xbal sticky end] generating SPD910.
  • SPD910 was digested with SacI (within IBI24) and Aval (within DEN at nucleotide 423) and ligated to an Aval-Sad fragment of DEN cDNA (nucleotides 424-1447 Mason et al. , 1987B) generating DEN4 encoding the carboxy-terminal 11 aa C, prM and amino-terminal 36% E.
  • Plasmid DEN23 containing DEN cDNA encoding the carboxy-terminal 55% NS2A and amino-terminal 28% NS2B (nucleotides 3745-4213, FIG. 20) (SEQ ID NO:53) was derived by cloning a Xbal-SphI fragment of DEN cDNA into Xbal-SphI digested IBI25.
  • Plasmid DEN20 containing DEN cDNA encoding the carboxy-terminal 55% NS2A, NS2B and amino-terminal 24 amino acids NS3 (nucleotides 3745-4563, FIG. 20) (SEQ ID NO:53) was derived by cloning a Xbal to EcoRI fragment of DEN cDNA into Xbal-EcoRI digested IBI25.
  • Oligonucleotide-directed mutagenesis was used to change potential vaccinia virus early transcription termination signals (Yuen et al., 1987) in the prM gene in DEN4 29 aa from the carboxy-terminus (nucleotides 822-828 TTTTTCT to TATTTCT) and 13 aa from the — oo—
  • Oligonucleotide-directed mutagenesis described above was used to insert an EaqI and EcoRI site at the carboxy-terminus of NS2A (nucleotide 4102) in plasmid DEN23 creating DEN24, to insert a Smal site and ATG 15 aa from the carboxy-terminus of E in DEN7 (nucleotide 2348) creating DEN10, to insert an EagI and Hindlll site at the carboxy- terminus of NS2B (nucleotide 4492) in plasmid DEN20 creating plasmid DEN21, and to replace nucleotides 60-67 in plasmid DEN15 with part of the vaccinia virus early/late H6 promoter (positions -1 to -21, Perkus et al., 1989) creating DEN16 (containing DEN nucleotides 20-59, EcoRV site to -1 of the H6 promoter and DEN nucleotides 68-1447
  • a Sacl-Xhol fragment from DEN7 (nucleotides 1447- 2579) was substituted for the corresponding region in DEN3 generating DEN19 containing DEN cDNA encoding the carboxy- terminal 64% E and amino-terminal 45% NS2A (nucleotides 1447-3745) with nucleotide 2467 present and the modified transcription termination signal (nucleotides 2448-2454) .
  • a Xhol-Xbal fragment from DEN19 (nucleotides 2579-3745) and a Xbal-Hindlll fragment from DEN24 (Xbal nucleotide 3745 DEN through Hindlll in IBI25) were ligated to Xhol-Hindlll digested IBI25 creating DEN25 containing DEN cDNA encoding the carboxy-terminal 82% NSl, NS2A and amino-terminal 28% NS2B (nucleotides 2579-4213) with a EaqI site at 4102, nucleotide 2467 present and mutagenized transcription termination signal (nucleotides 2448-2454) .
  • Xhol-Xbal fragment from DEN19 was ligated to Xhol (within IBI25) and Xbal (DEN nucleotide 3745) digested DEN21 creating DEN22 encoding the carboxy-terminal 82% NSl, NS2A, NS2B and amino-terminal 24 aa NS3 (nucleotides 2579- 4564) with nucleotide 2467 present, modified transcription termination signal (nucleotides 2448-2454) and EaqI site at 4492.
  • a Hindlll-PstI fragment of DEN16 (nucleotides 20- 59, EcoRV site to -1 of the H6 promoter and DEN nucleotides 68-494) was ligated to a Hindlll-PstI fragment from DEN47 (encoding the carboxy-terminal 83% prM and amino-terminal 36% of E nucleotides 494-1447 and plasmid origin of replication) generating DEN17 encoding C, prM and amino- terminal 36% E with part of the H6 promoter and EcoRV site preceding the amino-terminus of C.
  • a Hindlll-Bqlll fragment from DEN17 encoding the carboxy-terminal 13 aa C, prM and amino-terminal 36% E (nucleotides 370-1447) was ligated to annealed oligonucleotides SP111 and SP112 (containing a disabled Hindlll sticky end, EcoRV site to -1 of the H6 promoter, and DEN nucleotides 350-369 with a Bqlll sticky end) creating DEN33 encoding the EcoRV site to -1 of the H6 promoter, carboxy-terminal 20 aa C, prM and amino-terminal 36% E.
  • Smal-EagI digested pTP15 (Mason et al., 1991) was ligated to a Smal-SacI fragment from DEN4 encoding the carboxy-terminal 11 aa C, prM and amino-terminal 36% E (nucleotides 377-1447) and SacI-EagI fragment from DEN3 encoding the carboxy-terminal 64% E, NSl and amino-terminal 45% NS2A generating DENL.
  • a unique Smal site (located between the H6 promoter and ATG) was removed using oligonucleotide-directed double-strand break mutagenesis (Mandecki, 1986) creating DEN8VC in which the H6 promoter immediately preceded the ATG start codon.
  • nucleotides 2579-4102 was ligated to an XhoI-EagI fragment of DEN18 (containing the origin of replication, vaccinia sequences and DEN C prM, E and ' amino-terminal 18% NSl nucleotides 68-2579) generating DEN26.
  • a Sacl-Xhol fragment from DEN10 (nucleotides 1447- 2579) was substituted for the corresponding region in DEN3 generating DENll containing DEN cDNA encoding the carboxy- terminal 64% E, NSl and amino-terminal 45% NS2A with a Smal site and ATG 15 aa from the carboxy-terminus of E.
  • a Smal- EagI fragment from DENll (encoding the carboxy-terminal 15 aa E, NSl and amino-terminal 45% NS2A nucleotides 2348-3745) was ligated to Smal-EagI digested pTP15 generating DEN12.
  • a XhoI-EagI fragment from DEN22 (nucleotides 2579- 4492) was ligated to the XhoI-EagI fragment from DEN18 described above generating DEN27.
  • An EcoRV-PstI fragment from DEN12 (positions -21 to -1 H6 promoter DEN nucleotides 2348-3447 encoding 15aaE, NSl) was ligated to an EcoRV-PstI fragment from DEN27 (containing the origin of replication, vaccinia sequences, H6 promoter -21 to -124 and DEN cDNA encoding NS2A and NS2B) generating DEN31.
  • Oligonucleotides DEN 1 (SEQ ID NO:38), DEN 2 (SEQ ID NO:39), DEN9 (SEQ ID NO:40), DEN10 (SEQ ID NO:41), SP11 (SEQ ID NO:42), and SP112 (SEQ ID NO:
  • mice Groups of 3 week old mice were inoculated ip with 10 7 pfu vaccinia recombinants VP962, VP955, VP867, vP452
  • Table 12 shows that mice immunized twice with vP962 developed high levels of HAI antibodies, levels were equivalent to those obtained in animals immunized twice with Dengue type 1 Hawaii strain. Table 12. HAI antibody titers
  • DEN34 was digested with EcoRV (within the H6 promoter) and Hindlll within E (DEN nucleotide 2061; Mason et al., 1987b) and a 1733bp fragment (containing EcoRV to -1 H6 promoter, 20 aaC, prM and amino- terminal 77% E) was isolated.
  • DEN36 was digested with
  • NSl NSl
  • NS2A a canarypox donor plasmid (JEVCPC5) encoding 15aaC, prM, E.
  • the parental canarypox virus (Rentschler strain) is a vaccinal strain for canaries.
  • the vaccine strain was obtained from a wild type isolate and attenuated through more than 200 serial passages on chick embryo fibroblasts.
  • plaque purified canarypox isolate is designated
  • GCTTCCCGGGAATTCTAGCTAGCTAGTTT This replacement sequence contains Hindlll. Smal and EcoRI insertion sites followed by translation stops and a transcription termination signal recognized by vaccinia virus RNA polymerase (Yuen et al.,
  • RW145 (SEQ ID NO:60):
  • RW146 (SEQ ID N0J61):
  • Oligonucleotides A through E which overlap the translation initiation codon of the H6 promoter with the ATG of rabies G, were cloned into pUC9 as pRW737. Oligonucleotides A through E contain the H6 promoter, starting at Nrul. through the Hindlll site of rabies G followed by Bqlll. Sequences of oligonucleoties A through E are:
  • A (SEQ ID NO:62): CTGAAATTATTTCATTATCGCGATATCCGTTAAGTTT GTATCGTAATGGTTCCTCAGGCTCCTGTTTGT
  • AATTTCAG C (SEQ ID NO:64): ACCCCTTCTGGTTTTTCCGTTGTGTTTTGGGAAATT
  • Oligonucleotides A through E were kinased, annealed (95°C for 5 minutes, then cooled to room temperature) , and inserted between the PvuII sites of pUC9.
  • the resulting plasmid, pRW737 was cut with Hindlll and Bglll and used as a vector for the 1.6 kbp Hindlll-BqIII fragment of ptgl55PRO (Kieny et al., 1984) generating pRW739.
  • the ptgl55PRO Hindlll site is 86 bp downstream of the rabies G translation initiation codon.
  • Bqlll is downstream of the rabies G translation stop codon in ptgl55PRO.
  • pRW739 was partially cut with Nrul, completely cut with Bqlll, and a 1.7 kbp Nrul-Bqlll fragment, containing the 3* end of the H6 promoter previously described (Taylor et al., 1988a,b; Guo et al., 1989; Perkus et al., 1989) through the entire rabies G gene, was inserted between the Nrul and BamHI sites of pRW824. The resulting plasmid is designated pRW832. Insertion into pRW824 added the H6 promoter 5* of Nrul.
  • the pRW824 sequence of BamHI followed by Smal is: GGATCCCCGGG.
  • pRW824 is a plasmid that contains a nonpertinent gene linked precisely to the vaccinia virus H6 promoter. Digestion with Nrul and BamHI completely excised this nonpertinent gene. The 1.8 kbp pRW832 Smal fragment, containing H6 promoted rabies G, was inserted into the Smal of pRW831, to form plasmid pRW838.
  • Plasmid JEVL14VC containing JEV cDNA encoding 15 amino acids C, prM, E, NSl, NS2A in a vaccinia virus donor plasmid (FIG. 1) (nucleotides 337-4125, FIG.
  • JEVL14VC was digested with EclXI at the EaqI site following the T5AT, filled in with the Klenow fragment of DNA polymerase I and digested with SacI in JEV sequences (nucleotide 2124) generating a 2011 bp fragment.
  • the 1809 bp EcoRV-SacI, 2011 bp Sad-filled EclXI and 3202 bp EcpRV filled EcoRI fragments were ligated generating JEVCPl.
  • JEVCPl was transfected into ALVAC infected primary CEF cells to generate the canarypox recombinant VCP107 encoding 15 amino acids C, prM, E, NSl, NS2A (FIG. 18).
  • PrM E
  • a C5 insertion vector containing 1535 bp upstream of C5 polylinker containing Kpnl/Smal/Xbal and NotI sites and 404 bp of canarypox DNA (31 base pairs of C5 coding sequence and 473 bp of downstream sequence) was derived in the following manner.
  • a genomic library of canarypox DNA was constructed in the cosmid vector puK102 (Knauf et al.,
  • the 1535 bp upstream sequence was generated by PCR amplification (Engelke et al., 1988) using oligonucleotides C5A (SEQ ID NO:67) (5'-ATCATCGAATTCTGAATGTTAAATGTTATACTTTG- 3 « ) and C5B (SEQ ID NO:68) (5 » -GGGGGTACCTTTGAGAGTACCACTTCAG-
  • genomic canarypox DNA was digested with EcoRI (within oligoCSA) and cloned into EcoRI/Smal digested pUC8 generating C5LAB.
  • the 404 bp arm was generated by PCR amplification using oligonucleotides C5C (SEQ ID NO:69) (5'-GGGTCTAGAGCGGCCGCT TATAAAGATCTAAAATGCATAATTTC-3 ') and C5DA (SEQ ID NO:70) (5 1 - ATCATCCTGCAGGTATTCTAAACTAGGAATAGATG-3' . This fragment was — ./,o,,—
  • pC5L was digested within the polylinker with Asp.718 and Notl.
  • oligonucleotides CP26 SEQ ID NO:71
  • CP27 SEQ ID NO:72
  • CP26 SEQ ID NO:71
  • CP27 SEQ ID NO:72
  • a disabled Asp718 site translation stop codons in six reading frames
  • vaccinia early transcription termination signal Yuen and Moss, 1987
  • BamHI Kpnl Xhol Xbal Clal and Smal restriction sites vaccinia early transcription termination signal
  • translation stop codons in six reading frames generating plasmid C5LSP.
  • the early/late H6 vaccinia virus promoter (Guo et al., 1989; Perkus et al., 1989) was derived by PCR (Engelke et al., 1988) using pRW824 as template and oligonucleotides CP30
  • Plasmid VQH6C5LSP was digested within the H6 promoter with EcoRV and within the polylinker with Xbal and ligated to the 2065 bp fragment plus annealed oligonucleotides SP131 (SEQ ID NO:75) and SP132 (SEQ ID NO:76) (containing a SphI sticky end, T nucleotide completing the E coding region, translation stop, a vaccinia early transcription termination signal (AT5AT; Yuen and Moss, 1987) , a second translation stop, and Xbal , fi
  • JEVCP5 which encodes 15 amino acids C, prM and E under the control of the H6 promoter between C5 flanking arms. JEVCP5 can be transfected in
  • SP131 SEQ ID NO:75
  • SP132 SEQ ID NO:76
  • Primers for the 5' sequence were RG277 (SEQ ID NO:77) (5•-CAGTTGGTACCACT GGTATTTTATTTCAG-3 ') and RG278 (SEQ ID NO:78) (5'-TATCTGAATT CCTGCAGCCCGGGTTTTTATAGCTAATTAGTCAAATGTGAGTTAATATTAG-3') .
  • Primers for the 3' sequences were RG279 (SEQ ID NO:79) (5'TCGCTGAATTCGATATCAAGCTTATCGATTTTTATGACTAGTTAATC AAATAAAAAGCATACAAGC-3' ) and RG280 (SEQ ID NO:80) (5'-TTAT CGAGCTCTGTAACATCAGTATCTAAC-3 •) .
  • the primers were designed to include a multiple cloning site flanked by vaccinia transcriptional and translational termination signals. Also included at the 5'-end and 3'-end of the left arm and right arm were appropriate restriction sites (Asp718 and EcoRI for left arm and EcoRI and SacI for right arm) which enabled the two arms to ligate into Asp718/Sacl digested pBS-SK plasmid vector. The resultant plasmid was designated as pC3I.
  • a 908 bp fragment of canarypox DNA, immediately upstream of the C3 locus was obtained by digestion of plasmid pWW5 with Nsil and Sspl.
  • a 604 bp fragment of canarypox and DNA was obtained by digestion of plasmid pWW5 with Nsil and Sspl.
  • a 604 bp fragment of canarypox and DNA was obtained by digestion of plasmid pWW5 with Nsil and Sspl.
  • 24A-C (SEQ ID NO:83) was derived by PCR (Engelke et al., 1988) using plasmid pWW5 as template and oligonucleotides CP16 (SEQ ID NO:81) (5'- TCCGGTACCGCGGCCGCAGATATTTGTTAGCTTCTGC-3') and CP17 (SEQ ID NO:82) (5'-TCGCTCGAGTAGGATACCTACCTACTACCTACG-3') .
  • the 604 bp fragment was digested with Asp718 and Xhol (sites present at the 5' ends of oligonucleotides CP16 and CP17, respectively) and cloned into Asp718-XhoI digested and alkaline phosphatase treated IBI25 (International Biotechnologies, Inc., New Haven, CT) generating plasmid SPC3LA.
  • SPC3LA was digested within IBI25 with EcoRV and within canarypox DNA with Nsil, (nucleotide 536, FIG. 24A-C (SEQ ID NO:83)) and ligated to the 908 bp Nsil-Ssol fragment generating SPCPLAX which contains 1444 bp of canarypox DNA upstream of the C3 locus.
  • a 2178 bp Bglll-Styl fragment of canarypox DNA (nucleotides 3035-5212, FIG. 24A-C (SEQ ID NO:83)) was isolated from plasmids pXX4 (which contains a 6.5 kb Nsil fragment of canarypox DNA cloned into the PstI site of pBS- SK.
  • pXX4 which contains a 6.5 kb Nsil fragment of canarypox DNA cloned into the PstI site of pBS- SK.
  • a 279 bp fragment of canarypox DNA (nucleotides 5194-
  • FIG. 24A-C SEQ ID NO:83) was isolated by PCR (Engelke et al. , 1988) using plasmid pXX4 as template and oligonucleotides CP19 (SEQ ID NO:84) (5'-
  • TCGCTCGAGCTTTCTTGACAATAACATAG-3' TCGCTCGAGCTTTCTTGACAATAACATAG-3'
  • CP20 SEQ ID NO:85
  • the 279 bp fragment was digested with Xhol and SacI (sites present at the 5' ends of oligonucleotides CP19 and CP20, respectively) and cloned into Sacl-Xhol digested and alkaline phosphatase treated IBI25 generating plasmid SPC3RA.
  • pC3I was digested within the polylinker region with EcoRI and Clal, treated with alkaline phosphatase and ligated to kinased and annealed oligonucleotides CP12 (SEQ ID NO:86) and CP13 (SEQ ID NO:87) (containing an EcoRI sticky end, Xhol site, BamHI site and a sticky end compatible with Clal) generating plasmid SPCP3S.
  • SPCP3S was digested within the canarypox sequences downstream of the C3 locus with Styl (nucleotide 3035) and SacI (pBS-SK) and ligated to a 261 bp Bglll-SacI fragment from SPC3RA (nucleotides 5212-5472, FIG. 24A-C (SEQ ID NO:83)) and the 2178 bp Bqlll-Stvl fragment from pXX4 (nucleotides 3035-5212, FIG. 24A-C (SEQ ID NO:83)) generating plasmid CPRAL containing 2572 bp of canarypox DNA downstream of the C3 locus.
  • SPCP3S was digested within the canarypox sequences upstream of the C3 locus with Asp718 (in pBS-SK) and AccI (nucleotide 1435) and ligated to a 1436 bp Asp718-Accl fragment from SPCPLAX generating plasmid CPLAL containing 1457 bp of canarypox DNA upstream of the C3 locus.
  • CPLAL was digested within the canarypox sequences downstream of the C3 locus with Styl (nucleotide 3035) and SacI (in pBS-SK) and ligated to a 2438 bp Styl-SacI fragment from CPRAL generating plasmid CP3L containing 1457 bp of canarypox DNA upstream of the C3 locus, stop codons in six reading frames, early transcription termination signal, a polylinker region, early transcription termination signal, stop codons in six reading frames, and 2572 bp of canarypox DNA downstream of the C3 locus.
  • the early/late H6 vaccinia virus promoter (Guo et al., 1989; Perkus et al. , 1989) was derived by PCR (Engelke et al., 1988) using pRW838 as template and oligonucleotides CP21 (SEQ ID NO:88) (5'-TCGGGATCCGGGTTAATTAATTAGTTATTAGACAAG GTG-3 1 ) and CP22 (SEQ ID NO:89) (5'-TAGGAATTCCTCGAGTACGATACA AACTTAAGCGGATATCG-3 ') .
  • the PCR product was digested with
  • CP3L that was digested with BamHI and EcoRI in the polylinker generating plasmid VQH6CP3L.
  • CP12 (SEQ ID NO: 85) 5'-AATTCCTCGAGGGATCC -3'
  • CP13 SEQ ID NO:86) 3'- GGAGCTCCCTAGGGC-5'
  • ALVAC donor plasmid VQH6CP3L was digested within the polylinker with Xhol and Smal and ligated to a 3772 bp Xhol-Smal fragment from YF51 (nucleotides 419-4180 encoding YF 21 amino acids C, prM, E, NSl, NS2A) generating YF52.
  • the 6 bp corresponding to the unique Xhol site in UP52 were removed using oligonucleotide-directed double-strand break mutagenesis (Mandecki, 1986) creating YFCP3.
  • YFCP3 was transfected into ALVAC infected primary CEF cells to generate the canarypox recombinant VCP127 encoding 21 aa C, prM, E, NSl, NS2A (FIG. 19). Construction of C3 Insertion Vector Containing YFV 21 aa c, prM, E
  • YP52 was digested with Smal at the 3' end of the YF cDNA and Apal (YF nucleotide 1604) , a 8344 bp fragment isolated (containing the plasmid origin of replication, canarypox DNA and YF cDNA encoding 21 amino acids C, prM, and amino-terminal 57% E) and ligated to a Apal to Smal fragment from YF16 (nucleotides 1604-2452 containing the carboxy-terminal 43% E) generating YF54.
  • YFCP4 containing YF cDNA encoding 21 amino acids C, prM, and E.
  • YFCP4 can be transfected into ALVAC or ALVAC recombinant infected cells to generate a recombinant encoding YFV 21 aa C, prM, E.

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Abstract

On décrit un poxvirus recombiné, tel que le virus de la vaccine, le virus du pox aviaire et le virus du pox du canari, contenant de l'ADN étranger provenant d'un flavivirus, tel que le virus de l'encéphalite japonaise, le virus de la fièvre jaune et le virus de Dengue. Dans un mode préféré de réalisation, le poxvirus recombiné génère une particule extracellulaire contenant des protéines E et M du flavivirus capables d'induire des anticorps neutralisants et des anticorps inhibant l'hémagglutination et de produire une immunité protectrice contre l'infection à flavivirus. On décrit également un vaccin contenant le poxvirus recombiné et permettant d'induire une réaction immunitaire chez un animal hôte auquel on a inoculé ce vaccin.
PCT/US1991/005816 1990-08-15 1991-08-15 Vaccin a base de poxvirus recombine contre le flavivirus WO1992003545A1 (fr)

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AU87287/91A AU657711B2 (en) 1990-08-15 1991-08-15 Flavivirus recombinant poxvirus vaccine
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GB9303023A GB2269820B (en) 1990-08-15 1991-08-15 Recombinant pox virus encoding flaviviral structural proteins

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US07/714,687 US5514375A (en) 1990-08-15 1991-06-13 Flavivirus recombinant poxvirus vaccine
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DE19521705A1 (de) * 1995-06-14 1996-12-19 Immuno Ag Immunogenes Konstrukt, Verfahren zu dessen Herstellung und Verwendung als Vakzine
WO1996040933A1 (fr) * 1995-06-07 1996-12-19 The Government Of The United States Of America, Represented By The Secretary Department Of Health And Human Services Virus pdk-53 infectieux de la dengue 2 utilise comme vaccin quadrivalent
US5843742A (en) * 1994-12-16 1998-12-01 Avigen Incorporated Adeno-associated derived vector systems for gene delivery and integration into target cells
US5885808A (en) * 1992-11-04 1999-03-23 Imperial Cancer Research Technology Limited Adenovirus with modified binding moiety specific for the target cells
WO1999015692A2 (fr) * 1997-09-23 1999-04-01 Bavarian Nordic Research Institute A/S Antigenes du virus de la dengue et traitement de la dengue
US5925358A (en) * 1993-02-26 1999-07-20 Syntro Corporation Recombinant fowlpox viruses and uses thereof
WO1999063095A1 (fr) * 1998-06-04 1999-12-09 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Vaccins a base d'acides nucleiques destines a la prevention de l'infection par flavivirus
US6001369A (en) * 1993-02-26 1999-12-14 Syntro Corporation Recombinant fowlpox viruses and uses thereof
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US5744141A (en) 1998-04-28
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GB2269820B (en) 1995-03-29
JP3955315B2 (ja) 2007-08-08
AU657711B2 (en) 1995-03-23
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GB9303023D0 (en) 1993-12-01
US5744140A (en) 1998-04-28

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